title: Mechanisms of platelet-stimulated colon cancer invasion: role of clusterin and thrombospondin 1 in regulation of the P38MAPK-MMP-9 pathway.
abstract: Platelets have been implicated in colon cancer metastasis and prognosis but the underlying molecular mechanisms remain unclear. We evaluated the role of the different mitogen-activated protein kinase (MAPK) pathways in platelet-stimulated matrix metalloproteinase-9 (MMP-9) generation and colon cancer invasion. In addition, proteins released during platelet-tumour cell interactions were studied. For this purpose, interactions of Caco-2 and HT29 cells with platelets were studied using scanning electron microscopy, aggregometry, flow cytometry and cell invasion chambers. Quantitative PCR and zymography were used to study MMP-9 gene expression and activity, respectively, whereas western blot was used to study p38MAPK. Finally, the origin of proteins during platelet-cancer cell interactions was investigated using stable isotope labelling by amino acids in cell culture (SILAC)-based proteomics. We found that platelets promoted p38MAPK phosphorylation and MMP-9 up-regulation in both cell lines, with the subsequent cell-invasion-promoting effects. Pharmacological inhibition of p38MAPK led to a significant down-regulation of MMP-9 and colon cancer cell invasiveness. Also, p38MAPK-small interfering RNA abolished the induction of platelet-stimulated MMP-9. SILAC experiments demonstrated that thrombospondin 1 (TSP1) was released mainly from platelets and clusterin by both platelets and cancer cells. Finally, inhibition of TSP1 and clusterin abolished p38MAPK phosphorylation, MMP-9 activity and platelet-stimulated colon cancer invasion. Our results indicate that platelet-secreted TSP1 and clusterin promote the signal regulation of MMP-9 in platelet-induced colonic cancer invasion via a P38MAPK-regulated pathway. These findings are relevant to the development of therapeutic approaches to preventing and reducing tumour cell metastasis induced by colon adenocarcinoma.
authors.affiliation: Department of Pharmacology, School of Pharmacy and Pharmaceutical Sciences.
authors.forename: Aneta
authors.initials: A
authors.lastname: Radziwon-Balicka
authors.forename: Maria J
authors.initials: MJ
authors.lastname: Santos-Martinez
authors.forename: J Jose
authors.initials: JJ
authors.lastname: Corbalan
authors.forename: Shane
authors.initials: S
authors.lastname: O'Sullivan
authors.forename: Achim
authors.initials: A
authors.lastname: Treumann
authors.forename: John F
authors.initials: JF
authors.lastname: Gilmer
authors.forename: Marek W
authors.initials: MW
authors.lastname: Radomski
authors.forename: Carlos
authors.initials: C
authors.lastname: Medina
mesh_terms: D017209:Apoptosis; D051778:Basic Helix-Loop-Helix Leucine Zipper Transcription Factors / Q000235:genetics / Q000378:metabolism*; D001792:Blood Platelets / Q000378:metabolism*; D015153:Blotting, Western; D018938:Caco-2 Cells; D002465:Cell Movement; D049109:Cell Proliferation; D002853:Chromatography, Liquid; D051152:Clusterin / Q000235:genetics / Q000378:metabolism*; D003110:Colonic Neoplasms / Q000235:genetics / Q000378:metabolism / Q000473:pathology*; D005434:Flow Cytometry; D015972:Gene Expression Regulation, Neoplastic; D019073:HT29 Cells; D006801:Humans; D020780:Matrix Metalloproteinase 9 / Q000235:genetics / Q000378:metabolism*; D009361:Neoplasm Invasiveness; D010766:Phosphorylation; D040901:Proteomics; D012333:RNA, Messenger / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction; D053719:Tandem Mass Spectrometry; D048051:p38 Mitogen-Activated Protein Kinases / Q000235:genetics / Q000378:metabolism*
chemical_list: D051778:Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; C495242:CLU protein, human; D051152:Clusterin; D012333:RNA, Messenger; C481348:SPZ1 protein, human; D048051:p38 Mitogen-Activated Protein Kinases; C579270:MMP9 protein, human; D020780:Matrix Metalloproteinase 9